Fusion polypeptide inhibiting VEGF-C, VEGF-D and/or angiopoietin-2, and use thereof

ABSTRACT

A fusion polypeptide capable of binding simultaneously to angiopoietin 2, VEGF-C and VEGF-D; or capable of binding simultaneously to VEGF-C and VEGF-D, and methods for the preparation and use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No.10-2013-0085515 on Jul. 19, 2013 with the Korean Intellectual PropertyOffice, the entire disclosure of which is hereby incorporated byreference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 48,364 Bytes ASCII (Text) file named“716812_ST25.TXT,” created on Jul. 14, 2014.

BACKGROUND

1. Field

Provided is a fusion polypeptide capable of binding simultaneously toangiopoietin 2, VEGF-C and VEGF-D, preparation and use thereof. Further,provided is a fusion polypeptide capable of binding simultaneously toVEGF-C and VEGF-D, preparation and use thereof.

2. Description of the Related Art

In a higher organism, blood vessels and lymphatic ducts are formed byvasculogenesis and angiogenesis. Vasculogenesis is a process of formingnew blood vessels from hemopoietic precursor cells, which mainly occursonly in very limited situations such as development of embryo and fetus,uterine aging, proliferation of placenta, luteinization and woundhealing. To the contrary, angiogenesis is a process of forming new bloodvessels through proliferation, migration and differentiation of vascularendothelial cells, and excessive angiogenesis becomes a cause ofdiseases such as cancers, inflammatory diseases (psoriasis, arthritis,and the like), ophthalmic diseases (diabetic retinopathy), lymphaticproliferative diseases (atherosclerosis, and the like), lymphaticmetastasis, and neurodegenerative diseases.

Thus, fundamental treatment of excessive angiogenesis-related diseasesmay be inhibition of angiogenesis, but currently used angiogenesisinhibitors are generally prepared by an organic synthesis method, causeserious side-effects and the effects are not satisfactory. Particularly,an angiogenesis inhibitor that targets blood vessels supplying nutrientsto tumor while not directly acting on cancer cells is considered as oneof most promising anticancer therapies because it may avoid drugresistance of cancer cells.

VEGF (Vascular endothelial growth factor) is a representative vascularendothelial growth factor controlling formation and development of bloodvessels, and interacts with hematoblast, vascular endothelial precursorcells, and mature endothelial cells. In mammals, VEGF-A, B, C, D andP1GF are known as VEGF. The biological function of VEGF is mediatedthrough VEGF receptors VEGFR1, VEGFR2 and VEGFR3. VEGFR has anextracellular domain consisting of 7 immunoglobulin (Ig)-like domains; atransmembrane domain; and an intracellular domain (a regulatoryjuxtamembrane domain, an intracellular tyrosine kinase domain, severaltyrosine residues). VEGF binds to each specific receptor which is thenactivated. For example, VEGF-A binds to VEGFR1 and VEGFR2 to performcritical functions for growth, migration and survival of vascularendothelial cells, which are essential for vasculogenesis andangiogenesis. VEGF-B and P1GF bind exclusively to VEGFR1, and studies onthe function of VEGF-B in angiogenesis and blood vascular systemgeneration are being continued. To the contrary, VEGF-C and -D bindVEGFR2 and VEGFR3 which is then activated, thereby performing criticalfunctions for growth, migration and survival of lymphatic endothelialcells and formation and maintenance of lymphatic ducts.

Angiogenesis by VEGF plays important roles in growth, invasion andmetastasis of cancers. It was found that VEGF is overexpressed invarious tumors such as lung cancer, stomach cancer, renal cancer,bladder cancer, ovarian cancer and uterine cancer, and it was reportedthat cancer with highly expressed VEGF has a bad prognosis. Thus,studies on inhibition of growth or metastasis of tumors by inhibitingthe activity of VEGF or inhibiting the function of cell receptor VEGFR1or VEGFR2 are being actively progressed. A representative study employsthe VEGF trap, which is a water soluble decoy VEGF receptor manufacturedby combining domains of VEGFR1 and VEGFR2 on the surface of cells andhas high affinity to VEGF-A. Up to date, the antitumor effect of VEGFhas been verified, and it has been reported that the VEGF trap hassuperior antitumor effect compared to VEGF monoclonal antibodybevacizumab or VEGFR2 antibody DC101.

Meanwhile, Angiopoietin-2 (Ang2) is known as a factor relating to cancerangiogenesis and metastasis, as well as ocular diseases involvingabnormal vasculogenesis and rheumatoid arthritis. The biologicalfunction of angiopoietin-2 is activated by binding to receptor tyrosinekinase (RTK) Tie2. Tie2 has an extracellular domain consisting ofIg-like domain 1, Ig-like domain 2, 3 EGF-like domains, Ig-like domain3, and 3 fibronectin type-III domains; a transmembrane domain; and anintracellular tyrosine kinase domain. Among the extracellular subdomainsof Tie2, Ig-like domain 2 is essential for angiopoietin binding, andIg-like domains 1 and 3 are required for stable binding of angiopoietin.

The present disclosure inventors further progressed the concept of VEGFtrap, to develop fusion polypeptide that binds simultaneously to VEGF-C,VEGF-D and/or angiopoietin 2 to inhibit their intracellular signaltransduction, thus having potent effect of inhibiting proliferation andmetastasis of cancer cells and lymphangiogenesis,

SUMMARY

Provided is a fusion polypeptide capable of binding simultaneously toangiopoietin 2, VEGF-C and VEGF-D.

Also provided is a fusion polypeptide capable of binding simultaneouslyto VEGF-C and VEGF-D.

Further provided are nucleic acid molecules encoding the fusion peptide,recombinant vectors including the nucleic acid molecules and cellstransformed with the recombinant vector, and related methods ofculturing such cells.

It is another embodiment to provide a pharmaceutical compositionincluding the fusion polypeptide.

Yet further provided is a method for treating a neovascular diseases,inhibiting angiogenesis or lymphangiogenesis, or inhibitingproliferation and metastasis of cancer, comprising administering atherapeutically effective amount of the fusion polypeptide to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a fusion polypeptide (hereinafter, referredto as “V-body”) including Ig-like domain 2 (“R2-2”) that is VEGFR2extracellular domain, Ig-like domain 2 (“R3-2”) that is VEGFR3extracellular domain, and the Fc region of an immunoglobulin.

FIG. 2 is a diagram showing two fusion polypeptide called AV-bodies. Theleft hand fusion polypeptide (hereinafter referred to as “AV-body 1”)includes a Tie2 extracellular domain (“Tie2-ECD”), Ig-like domain 2(“R2-2”) and Ig-like domain 3 (“R2-3”) that are VEGFR2 extracellulardomains, and the Fc region of immunoglobulin. The right side fusionpolypeptide (hereinafter, referred to as “AV-body 2”) includes Tie2extracellular domain (“Tie2-ECD”), Ig-like domain 2 (“R2-2”) that isVEGFR2 extracellular domain, Ig-like domain 2 (“R3-2”) that is VEGFR3extracellular domain, and the Fc region of an immunoglobulin.

FIG. 3 is a series of three photographs of SDS-PAGE gels confirming theexpression of fusion polypeptides V-body, AV-body 1, and AV-body 2according to Example 2.

FIG. 4 is a graph showing the effects of three fusion polypeptides(V-body, AV-body 1, AV-body 2) on migration inhibition of lymphaticendothelial cells (LEC) due to VEGF-D.

FIG. 5 is a graph showing the effect of fusion polypeptide AV-body 2 onmigration inhibition of lymphatic endothelial cells (LEC) due to VEGF-C(“VC”) and Ang2 (“A2”).

FIG. 6 provides a graph (A) showing the effect of fusion polypeptideAV-body 2 for inhibiting proliferation of lymphatic endothelial cells(LEC) due to VEGF-C (“VC”) and Ang2 (“A2”), and a graph (B) showing theeffect of fusion polypeptide AV-body 2 for inhibiting proliferation ofvascular endothelial cells (VEC) due to VEGF-C (“VC”) and Ang2 (“A2”).

DETAILED DESCRIPTION

Provided is a fusion polypeptide capable of binding simultaneously toangiopoietin 2, VEGF-C and VEGF-D, comprising

a Tie2 extracellular domain;

a VEGFR2 extracellular domain, a VEGFR3 extracellular domain, or both;and

an Fc region of an immunoglobulin.

Also provided is a fusion polypeptide capable of binding simultaneouslyto VEGF-C and VEGF-D, including VEGFR2 extracellular domain, VEGFR3extracellular domain, and the Fc region of immunoglobulin.

In one embodiment, the Tie2 extracellular domain may include Ig-likedomain 1, Ig-like domain 2, and 3 EGF-like domains.

In another embodiment, the VEGFR2 extracellular domain may include oneor more selected from the group consisting of Ig-like domain 1, Ig-likedomain 2, and Ig-like domain 3.

In yet another embodiment, the VEGFR3 extracellular domain may includeone or more selected from the group consisting of Ig-like domain 1,Ig-like domain 2, and Ig-like domain 3.

Also provided is a fusion polypeptide including

a Tie2 extracellular domain comprising Ig-like domain 1, Ig-like domain2, and three EGF-like domains;

a VEGFR2 extracellular domain comprising Ig-like domain 2 and Ig-likedomain 3; and

the Fc region of an immunoglobulin.

Provided is a fusion polypeptide including

a Tie2 extracellular domain comprising Ig-like domain 1, Ig-like domain2, and three EGF-like domains;

a VEGFR2 extracellular domain comprising Ig-like domain 2 and a VEGFR3extracellular domain comprising Ig-like domain 2; and

the Fc region of an immunoglobulin.

Further provided is a fusion polypeptide including extracellular domainof Ig-like domain 2, VEGFR3 extracellular domain of Ig-like domain 2,and the Fc region of immunoglobulin.

Also provided is a fusion polypeptide including

a Tie2 extracellular domain comprising amino acid residues 1st to 345thof SEQ ID NO: 1; and

a VEGFR2 extracellular domain comprising amino acid residues 123th to326th of SEQ ID NO: 2; and

the Fc region of an immunoglobulin.

Also provided is a fusion polypeptide including

a Tie2 extracellular domain comprising amino acid residues 1st to 345thof SEQ ID NO: 1;

a VEGFR2 extracellular domain comprising amino acid residues 120th to220th SEQ ID NO: 2, and a VEGFR3 extracellular domain comprising aminoacid residues 136th to 226th of SEQ ID NO: 3; and

the Fc region of an immunoglobulin.

Also provided is a fusion polypeptide including amino acid sequence ofSEQ ID NO: 6 or SEQ ID NO: 7.

Also provided is a fusion polypeptide including a VEGFR2 extracellulardomain comprising amino acid residues 120th to 220th of SEQ ID NO: 2, aVEGFR3 extracellular domain comprising amino acid residues 136th to226th of SEQ ID NO: 3, and the Fc region of an immunoglobulin.

Also provided is a fusion polypeptide including a polypeptide consistingof amino acid sequence of SEQ ID NO: 4, and the Fc region ofimmunoglobulin.

Also provided is a nucleic acid molecule encoding a fusion polypeptideas described herein.

Also provided is a recombinant vector including the nucleic acidmolecule.

Also provided is a cell transformed with the recombinant vector.

Also provided is a method for preparing a fusion polypeptide, includingculturing cells transformed with the recombinant vector.

Also provided is a pharmaceutical composition for prevention ortreatment of neovascular diseases, including the fusion polypeptide.

In an embodiment, the neovascular disease may include cancer, diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,neovascular glaucoma, erythrosis, proliferative retinopathy, psoriasis,hemophilic arthropathy, capillary proliferation in atherosclerosisplaque, keloid, wound granulation, vascular adhesion, rheumatoidarthritis, osteoarthritis, autoimmune disease, crohn's disease,restenosis, atherosclerosis, intestinal adhesion, cat scratch disease,ulcer, liver cirrhosis, nephritis, diabetic nephropathy, malignantnephrosclerosis, thrombotic microangiopathy, organ transplant rejection,glomerulopathy, diabetes mellitus, inflammation, or neurodegenerativedisease.

Also provided is a method for treating neovascular diseases, includingadministering an effective amount of the fusion polypeptide to asubject.

Also provided is a method for inhibiting angiogenesis and/orlymphovasculogenesis, including administering an effective amount of thefusion polypeptide to a subject.

Also provided is a method for inhibiting proliferation and/or metastasisof cancer, including administering an effective amount of the fusionpolypeptide to a subject.

Also provided is a method for simultaneously inhibiting VEGF-C andVEGF-D, including administering an effective amount of the fusionpolypeptide to a subject.

Also provided is a method for simultaneously inhibiting angiopoietin 2,VEGF-C and VEGF-D, including administering an effective amount of thefusion polypeptide to a subject.

Also provided is a use of the fusion polypeptide for prevention ortreatment of neovascular diseases.

Also provided is a use of the fusion polypeptide in the preparation ofmedicine for prevention and/or treatment of neovascular diseases.

Hereinafter, the present disclosure will be explained in detail.

As used herein, the term “fusion polypeptide” refers to two or moredifferent polypeptides or proteins artificially connected together. Inone embodiment, in order to bind simultaneously to VEGF-C, VEGF-D and/orangiopoietin2 to inhibit signal transduction thereof, the fusionpolypeptide may consist of extracellular domain of each receptor VEGFR2,VEGFR3 and/or Tie2. In another embodiment, the fusion polypeptide mayinclude the Fc region of an immunoglobulin to increase in vivostability.

The fusion polypeptide may act as a chimeric decoy receptor. A decoyreceptor is a “fake” receptor that decoys a substrate and binds thereto,and aims to inhibit binding of a substrate to a true receptor. Thus, thefusion polypeptide may consist of minimum binding domains for bindingsimultaneously to VEGF-C, VEGF-D and/or angiopoietin 2, and inhibitsbinding of VEGF-C, VEGF-D and/or angiopoietin 2 to each receptor VEGFR2,VEGFR3, and Tie2, thereby acting as a therapeutic agent of neovasculardiseases that has potent effects of inhibiting proliferation andmetastasis of cancer cells, and lymphangiogenesis.

Tie2 has an extracellular domain comprising Ig-like domain 1, Ig-likedomain 2, 3 EGF-like domains, Ig-like domain 3, and 3 fibronectintype-III domains; a transmembrane domain; and an intracellular tyrosinekinase domain (Augustin H G et al., Nature Reviews Molecular CellBiology, 2009, 10, 165˜177). The extracellular domains of Tie2 may beused as a fusion polypeptide component, and preferably, among theextracellular domains of Tie2, minimum extracellular subdomains requiredfor binding to angiopoietin may be selected to constitute fusionpolypeptide.

In one embodiment, the Tie2 extracellular domains may include Ig-likedomain, Ig-like domain 2 and 3 EGF-like domains. These are believed tobe minimum parts found through 3-dimensional structure analysis toindependently stably exist while Tie2 binds to angiopoietin 2. The aminoacid sequence of Tie2 is known as GenBank Registration NO: AAH35514.2(SEQ ID NO: 1), and among them, the fusion polypeptide may include apolypeptide including amino acid residues 1st˜345th of SEQ ID NO: 1(e.g., SEQ ID NO: 5).

VEGFR2 and VEGFR3 have an extracellular domain comprising 7immunoglobulin (Ig)-like domains; a transmembrane domain; andintracellular domains (a regulatory juxtamembrane domain, amintracellular tyrosine kinase domain, several tyrosine residues). Theextracellular domains of VEGFR2 and VEGFR3 may be used as a component ofthe fusion polypeptide, and preferably, among the extracellular domainsof VEGFR2 and VEGFR3, minimum extracellular domains capable of bindingto VEGF-C and VEGF-D may be selected to constitute the fusionpolypeptide.

In another embodiment, the VEGFR2 extracellular domain may be one ormore selected from the group consisting of Ig-like domain 1, Ig-likedomain 2 and Ig-like domain 3, and the VEGFR3 extracellular domain maybe one or more selected from the group consisting of Ig-like domain 1,Ig-like domain 2 and Ig-like domain 3 (Ferrara N et al., NatureMedicine, 2003, 9, 669˜676).

In another embodiment, the amino acid sequence of VEGFR2 is known asGenBank Registration NO: AAH35514.2 (SEQ ID NO: 2), and among them, thefusion polypeptide may include a polypeptide including amino acidresidues 120th˜220th of SEQ ID NO: 2, or polypeptide including aminoacid residues 123th˜326th of SEQ ID NO: 2. The amino acid sequence ofVEGFR3 is known as GenBank Registration NO: NP_891555.2 (SEQ ID NO: 3)and among them, the fusion polypeptide may include a polypeptideincluding amino acid residues 136th˜226th of SEQ ID NO: 3.

And, the fusion polypeptide may include the Fc region of immunoglobulinat N-terminal or C-terminal as a component. The Fc region may be of animmunoglobulin class selected from the group consisting of IgA, IgD,IgE, IgG and IgM, and the Fc of IgG may be selected from homogeneousIgG1, IgG2, IgG3, and IgG4, and any allotypes thereof. It may include awhole or a part of CH2 and CH3 constant domains, but not limitedthereto.

The Fc region may function as various effectors to remove antigens, andthe functions may be largely divided as follows. First, the Fc region ofantibody binds to Fc receptor (Fc R) on the surface of effector cellsuch as macrophage or NK cell to promote phagocytosis or degradation,thereby removing cells containing or bound to antigen(antibody-dependent cell-mediated cytotoxicity (ADCC)). Second,complement cascade is activated by the Fc region of antibody to make ahole in cell membranes, thereby destructing pathogenic cells (complementdependent cytotoxicity (CDC)). In addition to effector functions,another important function of the Fc region is to increase bloodresidence time of antibody. FcRn (neonatal Fc receptor) existing invascular endothelial cells may bind between CH2 and CH3 of the Fc regionto prevent degradation of IgG thus prolonging blood half life.

In addition to these biological functions, the Fc region may be used asaffinity-tag for convenience of purification of fusion polypeptideexpressed in culture medium.

According to one embodiment, the fusion polypeptide may include (i) Tie2extracellular domains of Ig-like domain 1, Ig-like domain 2 and 3EGF-like domains; (ii) VEGFR2 extracellular domains of Ig-like domain 2and Ig-like domain 3; and (iii) the Fc region of immunoglobulin.

According to another embodiment, the fusion polypeptide may include (i)Tie2 extracellular domains of Ig-like domain 1, Ig-like domain 2 and 3EGF-like domains; (ii) VEGFR2 extracellular domain of Ig-like domain 2,and VEGFR3 extracellular domain of Ig-like domain 2, and (iii) the Fcregion of immunoglobulin.

According to yet another embodiment, the fusion polypeptide may include(i) polypeptide including 1st˜345th amino acid residues of Tie2; (ii)polypeptide including amino acid residues 123th˜326th of VEGFR2; and(iii) the Fc region of immunoglobulin.

According to yet another embodiment, the fusion polypeptide may include(i) polypeptide including 1st˜345th amino acid residues of Tie2; (ii)polypeptide including amino acid residues 120th˜220th of VEGFR2, andpolypeptide including amino acid residues 136th˜226th of VEGFR3; and(iii) the Fc region of immunoglobulin.

According to yet another embodiment, the fusion polypeptide may includeextracellular domain of Ig-like domain 2, VEGFR3 extracellular domain ofIg-like domain 2, and the Fc region of immunoglobulin.

According to yet another embodiment, the fusion polypeptide may includepolypeptide including amino acid residues 120th˜220th of VEGFR2,polypeptide including amino acid residues 136th˜226th of VEGFR3, and theFc region of an immunoglobulin.

According to yet another embodiment, the fusion polypeptide may includea polypeptide consisting of amino acid sequence of SEQ ID NO: 4, SEQ IDNO: 6 or SEQ ID NO: 7, and the Fc region of immunoglobulin.

The fusion polypeptide may further include a signal sequence so as tofacilitate extracellular secretion when culturing cells. And, eachpolypeptide constituting the fusion polypeptide may be directlyconnected each other, or connected through a linker, a spacer or aconnector such as a restriction enzyme recognition site. If connectedthrough a linker, it may not preferably decrease whole activity.

As used herein, the term “linker” refers to a peptide inserted betweenproteins so as to increase structural flexibility of there proteins toenhance the activity of each protein, when connecting extracellulardomains of each receptor to prepare fusion polypeptide. The kind of thelinker or the number of amino acid is not specifically limited as longas it may minimize immune reactions, but 1 to 20 amino acids arepreferable, and 1 to 5 amino acids is more preferable.

The fusion polypeptide may include variants of fusion polypeptide aswell as polypeptide having wild type amino acid sequence. The variant offusion polypeptide refers to protein having different sequence from wildtype amino acid sequence by deletion, insertion, non-conservative orconservative substitution of at least one amino acid residue, orcombination thereof. Amino acid exchange in protein and peptide thatdoes not generally change the activity of molecules is known in the art(H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979).

And, the fusion polypeptide may be modified by phosphorylation,sulfation, acrylation, glycosylation, methylation, farnesylation, andthe like. The variant or modified product is a functional equivalent tonatural protein exhibiting identical biological activity, but ifnecessary, the property of the polypeptide may be varied. In anembodiment, structural stability of protein to heat, pH, and the likemay be increased or the activity of protein may be increased byvariation and modification of amino acid sequence.

The fusion polypeptide may be chemically synthesized or produced by generecombination, and it may be produced by transforming host cells using arecombinant vector and separating and purifying expressed protein.

Accordingly, there are provided, for preparation of fusion polypeptide,a nucleic acid molecule coding the fusion polypeptide, a recombinantvector including the same, a cell transformed with the recombinantvector, and a method for preparing fusion polypeptide using the same.

Various vectors such as plasmid, virus, cosmid, and the like may be usedas the vector for preparing fusion polypeptide. A recombinant vectorincludes a cloning vector and an expression vector. A cloning vectorincludes an origin of replication, for example, an origin of replicationof plasmid, phage or cosmid, and it is replicon to which another DNAfragment may be attached to replicate the attached fragment. Anexpression vector was developed for use in protein synthesis, and thosecommonly used for expression of foreign protein in plants, animals ormicroorganisms may be used. The recombinant vector may be constructed byvarious methods known in the art.

A recombinant vector is a carrier in which foreign DNA fragment isinserted, and generally refers to a fragment of double stranded DNA. Therecombinant vector should be operably linked to transcription andtranslation regulatory sequence so as to increase expression level oftransformed gene in host cell. The recombinant vector is a geneticconstruct including operably linked essential regulatory element so asto express genetic insert in cells of a subject, and standardrecombinant DNA technology may be used to prepare the genetic construct.

The kind of the recombinant vector is not specifically limited as longas it may express target gene in various host cells of prokaryotic cellsand eukaryotic cells and produce target protein, but a vector that maymass-produce foreign protein of similar forms to natural protein whilepossessing a promoter exhibiting potent activity and strong expressionis preferable. A recombinant vector preferably includes at least, apromoter, an initiation codon, genes coding target protein, atermination codon, and a terminator. Besides, it may appropriatelyinclude DNA coding signal peptide, enhancer sequence, 5′ and 3′non-translated region of target gene, selection marker region, orreplicable unit, and the like.

The host cell that may be transformed with the recombinant vector mayinclude prokaryotic cells and eukaryotic cells, and host with high DNAintroduction efficiency and high expression efficiency of the introducedDNA may be commonly used. The prokaryotic cells may includeenterobacteria and strains such as E. coli JM109, E. coli BL21, E. coliRR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, strain ofthe genus Bacillus such as Bacillus subtillis, Bacillus thuringiensis,Salmonella typhimurium, Serratia marcescens, and various Pseudomonassp., and the eukaryotic cells may include yeast (Saccharomycecerevisiae), insect cells, plant cells and animal cells, for example,CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2,3T3, RIN and MDCK cell lines, and the like.

To prepare a transformant by introducing a recombinant vector into hostcell, any introduction method widely known in the art may be used. Forexample, if the host cell is a prokaryotic cell, a CaCl₂ method orelectroporation, and the like may be used, and if the host cell iseukaryotic cell, microinjection, calcium phosphate precipitation,electroporation, liposome-mediated transfection and gene bombardment,and the like may be used, but not limited thereto.

The selection of transformed host cells may be easily conducted by anymethods widely known in the art, using a phenotype expressed byselection marker. For example, if the selection marker is a specificantibiotics resistant gene, a transformant may be easily selected byculturing a transformant in a medium containing the antibiotics.

If transformant expressing the recombinant vector is cultured in anutrient medium, useful protein may be prepared and separated in a largequantity. The medium and culture conditions may be appropriatelyselected from those commonly used according to host cell. Whenculturing, conditions such as temperature, pH of medium and culturetime, and the like should be appropriately controlled so as to besuitable for growth and development of cells and mass production ofprotein.

Fusion polypeptide produced from transformed cell may be recovereddirectly from medium or as a lysis product of cells. In the case of amembrane-binding type, it may be isolated from a membrane using asuitable surfactant solution (for example, triton-X 100) or by enzymaticcleavage. Cells used for expression of fusion polypeptide may bedestructed by various physical or chemical means such a repetition offreeze-thaw, sonication, mechanical destruction or an agent fordegrading cells, and may be separated and purified by common biochemicalseparation method. For example, the separation method includeselectrophoresis, centrifugation, precipitation, dialysis, chromatography(ion exchange chromatography, affinity chromatography, immunoaffinitychromatography, reverse phase HPLC, gel permeation HPLC), isoelectricfocusing and various modified and combined methods thereof, but notlimited thereto.

There is provided a potent therapeutic agent for neovascular diseasesthat may bind simultaneously to VEGF-C and VEGF-D to inhibitintracellular signal transduction thereof, thereby inhibitingproliferation and metastasis of cancer cells and lymphogenesis, and toachieve this, there is provided a fusion polypeptide with increased invivo stability by combining extracellular domains of VEGF-C and-D-recognizing receptors VEGFR2 and/or VEGFR3 and fusing Fc thereto.Furthermore, there is provided a fusion polypeptide that also inhibitsthe function of angiopoietin 2 and thus has more potent effect ofinhibiting angiogenesis, by additionally introducing a part ofangiopoietin 2 receptor Tie2.

The fusion polypeptide may be a chimeric decoy receptor that may bindsimultaneously to VEGF-C, VEGF-D and/or angiopoietin 2, and it may beused for treatment of neovascular diseases caused by angiogenesis ofVEGF-C, VEGF-D and/or angiopoietin 2.

Thus, there is provided a pharmaceutical composition for prevention ortreatment of neovascular diseases, including the fusion polypeptide.

As used herein, the term “prevention” refers to all acts of inhibitingor delaying occurrence, diffusion or recurrence of cancer orangiogenesis-related diseases by administration of the composition, and“treatment” refers to all acts of improving or favorably modifyingsymptoms of the diseases by administration of the composition.

As used herein, the term “angiogenesis” refers to a process of newlyforming blood vessels, i.e., generating new blood vessels in cells,tissues or organs, “neovascular” refers to newly formed blood vesselsthrough angiogenesis, and “angiogenesis” and “neovascular” may beinterchangeably used in the present invention. In the present invention,“neovascular diseases” or “angiogenesis-related diseases” mean diseasescaused by abnormal progression of the angiogenesis.

The angiogenesis-related diseases that can be prevented or treated bythe composition may include cancer, inflammatory disease, ophthalmicdisease, lymphatic proliferative disease, lymphatic metastasis,neurodegenerative disease. More specifically, it may include cancer,diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma, erythrosis, proliferative retinopathy,psoriasis, hemophilic arthropathy, capillary proliferation inatherosclerosis plaque, keloid, wound granulation, vascular adhesion,rheumatoid arthritis, osteoarthritis, autoimmune disease, crohn'sdisease, restenosis, atherosclerosis, intestinal adhesion, cat scratchdisease, ulcer, liver cirrhosis, nephritis, diabetic nephropathy,malignant nephrosclerosis, thrombotic microangiopathy, organ transplantrejection, glomerulopathy, diabetes mellitus, inflammation, orneurodegenerative disease.

Cancers that may be prevented or treated by the composition may includeuterine cervical cancer, lung cancer, pancreatic cancer, non-small celllung cancer, liver cancer, colon cancer, bone cancer, skin cancer, headcancer, cervical cancer, skin melanoma, intraocular melanoma, uterinecancer, ovarian cancer, rectal cancer, liver cancer, cerebral tumor,bladder cancer, blood cancer, gastric cancer, anal cancer, colon cancer,breast cancer, fallopian tube carcinoma, endometrial carcinoma, vaginalcancer, vulvar cancer, Hodgkin's disease, esophageal cancer, smallintestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroidcancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer,penis cancer, prostate cancer, bladder cancer, renal cancer, ureteralcancer, renal cell carcinoma, renal pelvis carcinoma, central nervoussystem tumor, spinal cord tumor, brain stem glioma, pituitary adenoma,and the like. The pharmaceutical composition may effectively inhibitmigration of cancer cells, and thus, it may be useful for prevention andtreatment of cancer metastasis.

The pharmaceutical composition, if necessary, may further includepharmaceutically acceptable carriers, diluents and/or excipients incommonly used amounts. The pharmaceutically acceptable carrier mayinclude lactose, dextrose, sucrose, sorbitol, mannitol, starch, acaciarubber, calcium phosphate, alginate, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate,talc, magnesium stearate, and mineral oil, and the like, but is notlimited thereto. The pharmaceutical composition may further include alubricant, a wetting agent, a sweetener, a flavoring agent, anemulsifier, a suspension, a preservative, and the like, besides theabove ingredients.

The composition may be formulated into various forms including oraldosage forms such as powder, granule, tablet, capsule, suspension,emulsion, syrup, aerosol, and the like, injections such as a sterilizedsolution for injection, and the like, and it may be orally administeredor administered via various routes including intravenous,intraperitoneal, subcutaneous, rectal, local administration, and thelike.

The composition may further include known anticancer drugs orangiogenesis inhibitors in addition to fusion polypeptide as activeingredients, and may be combined with other therapies known to be usedfor treatment of these diseases. Other therapies may includechemotherapy, radiotherapy, hormonal therapy, bone marrowtransplantation, stem cell replacement therapy, other biologicaltherapy, immunotherapy, and the like, but are not limited thereto.

Specific examples of the anticancer drugs that may be included in thepharmaceutical composition may include DNA alkylating agents such asmechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide,ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin,busulfan, thiotepa, cisplatin and carboplatin; anti-cancer antibioticssuch as dactinomycin (actinomycin D), doxorubicin: adriamycin,daunorubicin, idarubicin, mitoxantrone, plicamycin, mitomycin and CBleomycin; and plant alkaloids such as vincristine, vinblastine,paclitaxel, docetaxel, etoposide, teniposide, topotecan and iridotecan,and the like, but are not limited thereto.

Specific examples of the angiogenesis inhibitors that may be included inthe pharmaceutical composition may include angiostatin (plasminogenfragment); anti-angiogenic antithrombin III; angiozyme; ABT-627; Bay12-9566; benefin; bevacizumab; BMS-275291; cartilage-derived inhibitor,(CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; combretastatinA-4; endostatin (collagen X VIII fragment); fibronectin fragment;Gro-beta; halofuginone; heparinase; heparin hexasaccharide fragment;HMV833; human chorionic gonadotropin (hCG); IM-862; interferonalpha/beta/gamma; interferon derived protein (IP-10); interleukin-12;Kringle 5 (plasminogen fragment); marimastat; dexamethasone;metalloproteinase inhibitor (TIMP); 2-methoxyestradiol; MMI 270 (CGS27023A); MoAb IMC-1C11; neovastat; NM-3; Panzem; PI-88; placentaribonuclease inhibitor; plasminogen activator inhibitor; plateletfactor-4 (PF4); prinomastat; prolactin 16 kD fragment;proliferin-related protein (PRP); PTK 787/ZK 222594; retinoidsorimastat; squalamine; SS 3304; SU 5416; SU6668; SU11248;tetrahydrocortisol-S; tetrathiomolybdate; Thalidomide; Thrombospondin)-1(TSP-1); TNP-470; transforming growth factor-beta (TGF-b);vasculostatin; vasostatin (calreticulin fragment); ZD6126; ZD6474;farnesyl transferase inhibitor (FTI); and bisphosphonate (for example,alendronate, etidronate, pamidronate, resedronate, ibandronate,zoledronate, olpadronate, icandronate or neridronate), and the like, butare not limited thereto.

There is provided a method for prevention or treatment ofangiogenesis-related diseases, including administering a therapeuticallyeffective amount of fusion polypeptide to a subject.

Further, there is provided a method for inhibiting angiogenesis and/orlymphangiogenesis, including administering an effective amount of fusionpolypeptide to a subject.

Further, there is provided a method for inhibiting proliferation and/ormetastasis of cancer, including administering an effective amount offusion polypeptide to a subject.

Further, there is provided a use of the fusion polypeptide forprevention and/or treatment of neovascular diseases, or use of thefusion polypeptide in the preparation of medicine for prevention and/ortreatment of neovascular diseases.

As used herein, the term “administration” refers to supplying a materialto a patient by any appropriate method, and the composition may beorally or parenterally administered via common routes capable ofreaching to a target tissue. The parenteral administration may includeintravenous injection, subcutaneous injection, intramuscular injection,intraperitoneal injection, endothelial administration, localadministration, intranasal administration, intrapulmonary administrationand intrarectal administration, and the like. For oral administration,oral compositions should be formulated so as to be protected fromdegradation in the stomach or active agent should be coated, becauseprotein or peptide is digested. And, the composition may be administeredby any devices capable of delivering the composition to target cells.

As used herein, the term ‘subject’ includes human, monkeys, cows,horses, sheep, pigs, chicken, turkeys, common quails, cats, dogs, mice,rabbits or Guinea pigs, but is not limited thereto. According to oneexample, it is mammals, and according to another example, it is humanbeing.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of fusion polypeptide effectivefor treating or preventing target diseases. The suitable administrationamount of the pharmaceutical composition may be variously prescribedaccording to factors such as a formulation method, administrationmethod, age, weight, gender and illness state of a patient, food,administration time, administration route, excretion rate and reactionsensitivity. Preferable administration amount of the composition may bein the range of 1 to 100 mg/kg for adults, but is not limited thereto.

The composition may be administered as a single therapeutic agent or incombination with other therapeutic agents, may be administeredsequentially or simultaneously with conventional therapeutic agents, andmay be administered once or multiple times. Considering these, it isimportant to administer an amount capable of obtaining maximum effectwith minimum amount, which may be easily determined by one of ordinaryknowledge in the art.

There is provided a fusion polypeptide that simultaneously inhibitsbinding to angiopoietin 2 performing an important function for aging andstabilization of new blood vessels, and VEGF-C and/or -D involved inlymphangiogenesis, and by blocking their signal transduction in cancercells through the activation of these receptors, effects of inhibitinggrowth of cancer tissue through angiogenesis and metastasis of tumorthrough lymphatic vessels may be anticipated.

One or more embodiments will now be described in further detail withreference to the following Examples. However, these examples are for theillustrative purposes only and are not intended to limit the scope ofthe invention.

Example 1 Construction of Fusion Polypeptide

For the construction of V-body, Ig-like domain 2 of VEGFR2 (R2-2, aminoacid residues 120th-220th) and Ig-like domain 2 of VEGFR3 (R3-2, aminoacid residues 136th-226th) (SEQ ID NO: 4) were connected, and the genesequence was optimized so as to be produced in human cells andsynthesized by Bioneer (Korea). Tie2 extracellular domain (Tie2-ECD)corresponds to 1st-345th (SEQ ID NO:5) in amino acid sequence of Tie2(GenBank: AAH35514.2; SEQ ID NO: 1), and the gene sequence followsBC035514.1. The amino acid sequence of VEGFR2 follows NP_002244.1 (SEQID NO: 2), the amino acid sequence of VEGFR-3 follows NP_891555.2 (SEQID NO: 3), and the gene sequences were optimized so as to be produced inhuman cells and synthesized by Bioneer. For the construction of AV-body1, Tie2-ECD, and Ig-like domains 2, 3 of VEGFR-2 (R2-2, R2-3, amino acidresidues 123th-326th) were sequentially connected (SEQ ID NO: 6), andfor the construction of AV-body 2, Tie2-ECD, R2-2, and R3-2 weresequentially connected (SEQ ID NO: 7).

The prepared nucleic acid fragments were respectively subcloned in apFUSE-hIgG1-Fc1 vector (InvivoGen) that is constructed so as to fuse theFc fragment of human IgG1 to C-terminus of target protein. V-body wasconstructed by subcloning the nucleic acid fragments of R2-2 and R3-2 inthe vector using an EcoRI/XhoI restriction enzyme (NEB), and AV-body 1was constructed by first subcloning the nucleic acid fragments of R2-2and R2-3 in the vector using an EcoRI/XhoI restriction enzyme, and then,sequentially subcloning the nucleic acid fragment of Tie2-ECD using anAgeI/EcoRI restriction enzyme. AV-body 2 was constructed by subcloningthe nucleic acid fragment of Tie2-ECD in the above manufactured V-bodyconstruct using an AgeI/EcoRI restriction enzyme.

Example 2 Expression and Purification of Fusion Polypeptide

A nucleic acid molecule coding the fusion polypeptide manufactured inExample 1 was transfected into 293-F cells (Invitrogen) using a Maxtransfection reagent (Invitrogen). The cells were cultured by agitatingat 37° C., 8% CO₂, 130 rpm in serum-free 293-F expression medium(Invitrogen), and on the fifth day, the culture solution was recovered.From the culture solution obtained by centrifugation of the culturedcells, Fc-fused fusion polypeptide was selectively separated usingMabSelectSuRe column (GE Healthcare), and eluted with a 20 mM sodiumcitrate (pH 3.0) solution, and then, neutralized using a 1 M Tris (pH9.0) solution. The separated and purified fusion polypeptides wererespectively quantified using NanoDrop (Thermo Scientific), and the sizeand purity were confirmed under reducing, non-reducing conditionsthrough SDS-PAGE (FIG. 3).

Example 3 Measurement of Binding Capacity of Fusion Polypeptide to Ang2,VEGF-C, VEGF-D

To measure binding capacities of fusion polypeptides to each target,ELISA (Enzyme-linked immunosorbent assay) was conducted. On a 96-wellMaxiSorp™ flat-bottom plate (Nunc), 50 ul of human VEGF-C, VEGF-D, orAng2 (R&D Systems) were coated in the concentration of 5 ug/ul. Andthen, the plate was washed 5 times with PBS containing 0.05% Tween-20,and then, blocked with PBS containing 1% BSA at room temperature for 2hours. 50 ul of each fusion polypeptide was put in each well accordingto concentration, and then, the plate was incubated at room temperaturefor 2 hours. And then, it was washed 5 times with PBS containing 0.05%Tween-20, and then, HRP-conjugated anti-human Fc antibody (Sigma) wasdiluted to 1:5,000 with PBS containing 1% BSA and reacted at roomtemperature for 1 hour, and washed 5 times with PBS containing 0.1%Tween-20. Finally, to each well of the plate, 50 μl of TMB substrate(SIGMA) was added to cause color reaction, and then, the reaction wasstopped with 50 μl of a 5N H₂SO₄ solution, and OD450 value was measuredon a plate reader (Molecular Devices). Thereby, binding capacities(K_(D)) of the manufactured fusion polypeptides to human VEGF-C, VEGF-Dor Ang2 protein were measured. The results are described in thefollowing Table 1. As shown in Table 1, V-body binds to VEGF-C andVEGF-D but does not binds to Ang2, whereas both AV-body 1 and AV-body-2bind simultaneously to Ang2, VEGF-C and VEGF-D.

TABLE 1 Name Ang2 (K_(D), nM) VEGF-C (K_(D), nM) VEGF-D (K_(D), nM)V-body — 100 100 AV-body 1 45 130 170 AV-body 2 20 34 42

Example 4 Effect of Inhibiting Cell Migration of Fusion Polypeptide

The migration of lymphatic endothelial cells was measured usingxCelligence RTCA (Realtime cell analyzer) of GE Healthcare. RTCA isnon-invasive cell monitoring system capable of confirming change incells by measuring impedance in real-time. To conduct cell migrationassay, CIM-plate16 (GE Healthcare) consisting of a lower chamber and anupper chamber is used wherein microelectrodes for measuring impedanceare arranged in the upper chamber, and if cells seeded in the chambermigrate through micro-holes, they are attached to the microelectrode,thus allowing confirmation of migration of cells, which was representedby migration index. Lymphatic endothelial cells (P5-7) that were grownin EGM2-MV medium was grown for 6 hours in EBM medium to which 1% FBS isadded. Into each well of the lower chamber of CIM-plate16, 2 ug/mlVEGF-D and fusion polypeptide were put in EBM medium containing 1% FBS,and then, assembled with the upper chamber coated with fibronectin. 30ul of serum-free EBM medium was introduced in the upper chamber, andthen, put in an incubator for 1 hour for equilibration between the plateand the medium, a CIM-plate was installed in a device station in theincubator, and background value was measured. Lymphatic endothelialcells resuspended in serum-free medium were seeded at 40,000˜60,000cells/well, left to settle down for 15 minutes, and then, installed in adevice to measure cell migration in real-time. The migration degree ofcells was represented by Migration index, and it was confirmed that allthe three fusion polypeptides inhibit migration of lymphatic endothelialcells by VEGF-D (FIG. 4).

It was also confirmed that if VEGF-C and Ang2 are treated together bythe same experiment method as above, migration of lymphatic endothelialcells are more increased than single treatment group of each growthfactor, and that AV-Body 2 inhibit migration of lymphatic endothelialcells by the combined effect of VEGF-C and Ang2 (FIG. 5).

Example 5 Inhibition Effect of Cell Proliferation of Fusion Polypeptide

Proliferation of vascular and lymphatic endothelial cells were measuredusing Cell counting kit-8 (Dojindo Molecular Technology). P3˜P8 vascularendothelial cells or lymphatic endothelial cells were put in a Collagencoated 96 well plate (BD Bioscience) at 3,000˜5,000 cells/well, and thencultured. And, 2 ug/ml Ang2 and 1 ug/ml VEGF-C were sequentially orsimultaneously mixed with serum-free medium, and then, fusionpolypeptide AV-Body 2 was introduced into the medium. After removing theculture solution of 96-well plate, the plate was washed with PBS, andthen, medium containing growth factor and fusion polypeptide (AV-Body 2)was added, and cultured for 3 days. For CCK-8 assay, 10 ul of thesolution was added and cultured for 1 hour, and then, absorbance wasmeasured at 450 nm using a Microplate reader (Perkin Elmer). As theresult, as shown in FIG. 6, it was confirmed that in both of vascularendothelial cells and lymphatic endothelial cells, cell proliferationwas higher in combined treatment group than in each single treatmentgroup of VEGF-C and Ang2, and that cell proliferation by VEGF-C and Ang2was inhibited by AV-Body 2.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A fusion polypeptide capable of bindingsimultaneously to angiopoietin 2 (ANG2), Vascular endothelial growthfactor C (VEGF-C), and Vascular endothelial growth factor D (VEGF-D),the fusion polypeptide comprising (a) a Tie2 extracellular domaincomprising Ig-like domain 1, Ig-like domain 2, and three EGF-likedomains; (b) a Vascular endothelial growth factor receptor 2 (VEGFR2)extracellular domain comprising one or more domains selected from thegroup consisting of Ig-like domain 1, Ig-like domain 2, and Ig-likedomain 3; or a combination of the VEGFR2 extracellular domain and aVascular endothelial growth factor receptor 3 (VEGFR3) extracellulardomain comprising one or more domains selected from the group consistingof Ig-like domain 1, Ig-like domain 2, and Ig-like domain 3; and (c) anFc region of an immunoglobulin.
 2. The fusion polypeptide according toclaim 1, wherein the fusion polypeptide comprises a Tie2 extracellulardomain comprising Ig-like domain 1, Ig-like domain 2, and three EGF-likedomains; a VEGFR2 extracellular domain comprising Ig-like domain 2 andIg-like domain 3; and an Fc region of an immunoglobulin.
 3. The fusionpolypeptide according to claim 1, wherein the fusion polypeptidecomprises a Tie2 extracellular domain comprising Ig-like domain 1,Ig-like domain 2, and three EGF-like domains; a VEGFR2 extracellulardomain comprising Ig-like domain 2 and a VEGFR3 extracellular domaincomprising Ig-like domain 2; and the Fc region of an immunoglobulin. 4.The fusion polypeptide according to claim 1, wherein the fusionpolypeptide comprises a Tie2 extracellular domain comprising amino acidresidues 1-345 of SEQ ID NO: 1; and a VEGFR2 extracellular domaincomprising amino acid residues 123-326 of SEQ ID NO: 2; and the Fcregion of an immunoglobulin.
 5. The fusion polypeptide according toclaim 1, wherein the fusion polypeptide comprises a Tie2 extracellulardomain comprising amino acid residues 1-345 of SEQ ID NO: 1; a VEGFR2extracellular domain comprising amino acid residues 120-220 of SEQ IDNO: 2, and a VEGFR3 extracellular domain comprising amino acid residues136-226 of SEQ ID NO: 3; and the Fc region of an immunoglobulin.
 6. Thefusion polypeptide according to claim 1, wherein the fusion polypeptidecomprises a polypeptide consisting of the amino acid sequence of SEQ IDNO: 6 or SEQ ID NO: 7, and the Fc region of immunoglobulin.
 7. Apharmaceutical composition comprising the fusion polypeptide of claim 1.8. A method for treating a neovascular disease, inhibiting angiogenesisor lymphangiogenesis, or inhibiting proliferation and metastasis ofcancer in a subject in need thereof, comprising administering atherapeutically effective amount of the fusion polypeptide of claim 1 tothe subject.